The goal of this project is to create a novel siRNA delivery nanotechnology capable of treating and preventing the recurrence of glioblastoma, the most malignant primary human brain cancer. There is increasing evidence that brain tumor recurrence is due to the presence of brain tumor initiating cells (BTICs). These cells are believed to be able to survive conventional treatments and to be able to migrate away from the primary tumor site and form new tumors. RNA interference (RNAi), a natural cellular process that can prevent the expression of genes in a sequence-specific manner, can be induced by the introduction of short interfering RNA (siRNA) into the cells. Using siRNA to turn off the genes that allow BTICs to survive treatment, to migrate, and to form new tumors has the potential to prevent tumor recurrence following treatment. However, siRNA delivery is challenging. Viral siRNA delivery has many potential problems such as tumorigenicity and immunogenicity, and is generally limited to carrying one type of siRNA, as multiple doses could increase the risks of using them in patients. We have been able to synthesize a novel, bioreducible poly(beta-amino ester) (PBAE)-based nanoparticle capable of safe and effective delivery of siRNA to primary human glioblastoma cells. We have also shown that we can achieve near complete gene knockdown of a fluorescent marker gene using only a fraction of the siRNA that we can load into the nanoparticles. We have also completed in vitro work that suggests that these nanoparticles preferentially induce RNAi in BTICs and not in healthy human brain cells. We believe that we can even further optimize this delivery in a way that will enable delivery of multiple siRNAs simultaneously, and that we can determine the biological mechanism behind tumor selectivity. Based on our preliminary work, we hypothesize that we will be able to use these PBAE-based nanoparticles to codeliver multiple siRNAs within the same nanoparticle with the goal of simultaneously shutting down several biochemical pathways involved in tumor migration and growth. We hypothesize that this combinatorial approach will result in a robust phenotypic change that will prevent tumor recurrence and result in prolonged survival in a mouse model of brain cancer. In Specific Aim 1 we will engineer PBAE nanoparticle chemistry to test our hypothesis that we can further optimize siRNA delivery. In Specific Aim 2 we will determine the mechanism that enables tumor-specific delivery, and test the hypothesis that we can simultaneously and robustly knockdown the expression of multiple genes that promote tumor survival and prevent recurrence. Finally, in Specific Aim 3 we will examine the phenotypic change caused by knocking down the targeted genes both in vitro and in vivo to test the hypothesis that combinatorial, nanoparticle-mediated gene knockdown will slow tumor cell growth and tumor recurrence. This work will allow us to optimize and characterize a combinatorial, tumor-specific technology for the delivery of brain cancer therapeutics.